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Maintenance of Golgi structure and function depends on the integrity of ER export.

Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J - J. Cell Biol. (2001)

Bottom Line: We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model.A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region.These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.

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BFA remnants localize to ER exit sites. (A) ImmunoEM showing distribution of either Sec13–YFP or p58–GFP in NRK stable cell lines either left untreated or treated with BFA. Sec13–YFP labels membranes closely associated with VTCs, whereas p58–GFP is found both on the clusters and in surrounding ER membranes. (B) Characterization by light microscopy of cells treated with BFA. Sec13–YFP, transfected ± p58–CFP, and GRASP65–GFP stable NRKs were treated with BFA and then either visualized directly or fixed and stained for endogenous GM130. Arrows show colocalizing puncta, and the area inside the white box is enlarged in the inset. In untreated cells, endogenous GM130 localizes only to the Golgi apparatus (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A) 130 nm; (B) 5 μm.
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fig4: BFA remnants localize to ER exit sites. (A) ImmunoEM showing distribution of either Sec13–YFP or p58–GFP in NRK stable cell lines either left untreated or treated with BFA. Sec13–YFP labels membranes closely associated with VTCs, whereas p58–GFP is found both on the clusters and in surrounding ER membranes. (B) Characterization by light microscopy of cells treated with BFA. Sec13–YFP, transfected ± p58–CFP, and GRASP65–GFP stable NRKs were treated with BFA and then either visualized directly or fixed and stained for endogenous GM130. Arrows show colocalizing puncta, and the area inside the white box is enlarged in the inset. In untreated cells, endogenous GM130 localizes only to the Golgi apparatus (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A) 130 nm; (B) 5 μm.

Mentions: One potential location that p58 and Golgi matrix proteins could relocate to in BFA-treated cells is ER exit sites, since BFA is not thought to directly perturb COPII-dependent ER export machinery (Orci et al., 1993; Lippincott-Schwartz et al., 2000). A marker for these sites is Sec13–YFP (Hammond and Glick, 2000), which is a component of the COPII coat that mediates vesicle budding from zones of transitional smooth ER (i.e., ER exit sites) (Barlowe et al., 1994; Kuge et al., 1994). BFA did not disturb the overall distribution of Sec13–YFP (Fig. 3 A), which localized to punctate structures widely distributed in the cytoplasm that underwent little long-range motion (Hammond and Glick, 2000). At the EM level, these structures showed no apparent changes in morphology as a result of BFA treatment. In both BFA-treated and untreated cells, immunogold-labeled Sec13 was localized to the transitional ER that was adjacent to vesicular tubular clusters (VTCs) (Fig. 4 A).


Maintenance of Golgi structure and function depends on the integrity of ER export.

Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J - J. Cell Biol. (2001)

BFA remnants localize to ER exit sites. (A) ImmunoEM showing distribution of either Sec13–YFP or p58–GFP in NRK stable cell lines either left untreated or treated with BFA. Sec13–YFP labels membranes closely associated with VTCs, whereas p58–GFP is found both on the clusters and in surrounding ER membranes. (B) Characterization by light microscopy of cells treated with BFA. Sec13–YFP, transfected ± p58–CFP, and GRASP65–GFP stable NRKs were treated with BFA and then either visualized directly or fixed and stained for endogenous GM130. Arrows show colocalizing puncta, and the area inside the white box is enlarged in the inset. In untreated cells, endogenous GM130 localizes only to the Golgi apparatus (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A) 130 nm; (B) 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198855&req=5

fig4: BFA remnants localize to ER exit sites. (A) ImmunoEM showing distribution of either Sec13–YFP or p58–GFP in NRK stable cell lines either left untreated or treated with BFA. Sec13–YFP labels membranes closely associated with VTCs, whereas p58–GFP is found both on the clusters and in surrounding ER membranes. (B) Characterization by light microscopy of cells treated with BFA. Sec13–YFP, transfected ± p58–CFP, and GRASP65–GFP stable NRKs were treated with BFA and then either visualized directly or fixed and stained for endogenous GM130. Arrows show colocalizing puncta, and the area inside the white box is enlarged in the inset. In untreated cells, endogenous GM130 localizes only to the Golgi apparatus (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A) 130 nm; (B) 5 μm.
Mentions: One potential location that p58 and Golgi matrix proteins could relocate to in BFA-treated cells is ER exit sites, since BFA is not thought to directly perturb COPII-dependent ER export machinery (Orci et al., 1993; Lippincott-Schwartz et al., 2000). A marker for these sites is Sec13–YFP (Hammond and Glick, 2000), which is a component of the COPII coat that mediates vesicle budding from zones of transitional smooth ER (i.e., ER exit sites) (Barlowe et al., 1994; Kuge et al., 1994). BFA did not disturb the overall distribution of Sec13–YFP (Fig. 3 A), which localized to punctate structures widely distributed in the cytoplasm that underwent little long-range motion (Hammond and Glick, 2000). At the EM level, these structures showed no apparent changes in morphology as a result of BFA treatment. In both BFA-treated and untreated cells, immunogold-labeled Sec13 was localized to the transitional ER that was adjacent to vesicular tubular clusters (VTCs) (Fig. 4 A).

Bottom Line: We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model.A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region.These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.

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